Oilseeds such as tree nuts, peanut, soybean, and sesame are common allergenic foods that collectively affect &gt;3% of the US population. Legumin, a member of the cupin superfamily, is a major storage protein and a recognized allergen in oilseeds. Cupins, in general, are structurally conserved and stable against enzymatic proteolysis. The objective of this study was to analyze cross-reactivity and in vitro pepsin digestibility of legumins from different oilseeds. Whole proteins were extracted from defatted almond, Brazil nut, cashew, pistachio, soybean, and sesame flours. Legumins were purified using low-pressure liquid chromatography. Purified legumins were subjected to in vitro pepsin digestion at 800 unit/mg protein, 37°C, pH 3.0 for 1, 5, 15, 60, and 120 min. Immunoreactivity and cross-reactivity of the legumins were assessed by immunoblotting using seed-specific rabbit antisera and oilseed-allergic patients’ plasma IgE as detection antibodies. Despite low sequence identity (32-50%), the tested legumins were cross-reactive with each other in their native, denatured, and reduced forms. Sesame legumin exhibited the strongest cross-reactivity, while soy legumin the weakest. The basic subunit of the legumins was more cross-reactive than the acidic subunit except for almond legumin. Homology modeling revealed the greatest variation in immunodominant regions on soy legumin when compared to the other legumins. In vitro pepsin digestion under the experimental conditions suggested that legumins were stable against low pH and pepsin digestion. Reactivity of the digests to allergic patients’ IgE retained for at least 15 min during digestion, while reactivity to rabbit antisera lasted for 120 min. Considerable protein aggregation was observed throughout the digestion, and disulfide linkages remained intact. The persistence of disulfide bonds and acid-induced aggregation were likely the primary reasons for the stability of the legumins during digestion. Overall, our results suggested that legumins from taxonomically different seeds were cross-reactive and resistant to in vitro pepsin digestion.

Cross-reactivity and in vitro pepsin digestibility of allergenic oilseed legumins

ACS Fall 2020 Virtual Meeting

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